The incidence and mortality rates of malignant melanoma continue to rise dramatically throughout the world. In the United States, it is estimated that one in 90 Americans will develop melanoma by the year 2000. Melanoma is one of the most feared neoplasms because of the high mortality associated with metastatic involvement.
Malignant melanoma is commonly found in early stages in the form of a skin lesion. The lesion often results from the transformation of a preexisting nevus or discolored patch of skin containing aggregates of melanocytes. The best prognostic factor for determining the presence of metastatic disease is the depth of invasion of the primary lesion. Lesions with a depth greater than 0.8 mm have an increased risk for metastases. Melanomas usually metastasize first via the lymphatic system, with involvement of regional nodes, and then via blood vessels, with dissemination to subcutaneous tissue and to the liver, lungs, and brain. The presence of regional lymph node metastasis is predictive of a poor prognosis.
Malignant melanomas arise from melanocytes and can be pigmented (melanotic) due to accumulation of melanin, which imparts a dark color to these lesions. Due to the fact that some melanocytes may be less well-differentiated and therefore produce little or no melanin, these malignant lesions may also be nonpigmented or amelanotic.
Melanins are pigments responsible for the dark color of skin, hair, feathers, fur, insect cuticle, the choroid coat of the eye, and the substantia nigra of the brain. Melanins are also found in plants, fungi, bacteria, and pathological human urine where they can be an indication of melanotic tumors. These pigments are synthesized into the skin by melanocytes; they have a broad absorption spectrum from 250 nm-1,200 nm and are sometimes referred to as providing a "light absorbing mantle." Eumelanins (sepiomelanin, melanoma-melanin) and phaeomelanins are found in the animal kingdom, the latter group being lighter in color. Allomelanins are most often present in bacteria and plants. The chemical units that predominate in these melanins are indole-related, formed from tyrosine and dopa precursors.
Production of melanin is termed melanogenesis and is strictly compartmentalized to melanosomes, highly specialized organelles of melanocytes. Through dendritic extensions of the melanocyte, melanins are transferred to keratinocytes, resulting in pigmentation of the skin.
Physicians currently use CT scans, radionuclide bone scans, liver and spleen scintigraphy, and magnetic resonance imaging to evaluate the extent of melanoma. Usually these tests are conducted when specific symptoms require investigation. However, these diagnostic modalities are not specific for melanoma; biopsy is currently the only recognized definitive means of identifying the cause of a lesion.
Melanoma tumor-associated antigens expressed by melanoma cells have been used as a target for radioimmunoscintigraphy with radiolabeled antibodies. Radiolabeled monoclonal antibodies having specificity for particular cell types can identify the cellular composition of intact lesions. However, antigens expressed on the surface of melanoma cells are only somewhat melanoma-specific. Nonspecific localization of antibody conjugates in liver, spleen, and bone marrow is a significant problem in image interpretation. In addition, micrometastatic lesions are not able to be localized.
Sentinel nodes, the lymph nodes nearest the site of the primary melanoma tumor on the direct lymph drainage pathway, are identified by injecting patent blue-V or isosulfan intradermally at the site of the primary melanoma. If tumor is found in the sentinel node, then a full lymphadenectomy is carried out. Lymphatic channels are visualized during dissection and traced to the sentinel lymph node. A regional lymph node basin can be identified by cutaneous lymphoscintigraphy with technetium-labeled dextran in patients whose melanoma has an ambiguous drainage route.
Early detection of melanoma is highly desirable for patient prognosis. Circulating melanoma cells can be detected by cytometric methods and by using reverse transcriptase-polymerase chain reaction to detect tyrosinase messenger ribonucleic acid which is correlated with circulating melanoma cells. Melanin metabolites can be detected by HPLC, histology, or by immunohistochemical techniques. ARUP Laboratories (Salt Lake City, Utah) appears to provide a colorimetric test for melanin in urine (www.arup-lab.com/ug/ugar1399.htm, updated Apr. 1, 1997), although the reagent used is not specified.
A solution of sodium nitroferricyanide, also known as Thormahlin reagent, has been used by clinicians to detect urinary melanin for medical diagnostic purposes, such as for detection of melanoma. Melanin is detected by observance of a color change after the addition of sodium nitroferricyanide to an aliquot of urine. Depending upon the levels of melanin present in the sample, the mixture will change from a normal straw color to green, blue, brown, or black.
Demelanizing test samples of urine so that melanin does not interfere with testing for marijuana is reported by U.S. Pat. No. 4,843,020. Reagents such as sodium nitroferricyanide, acidified ferric chloride in water, hydroquinone (p-dihydroxybenzene), monobenzone (monobenzyl ether of hydroquinone), an ammoniacal silver nitrate solution in water, or certain mono- and dihydroxybenzene derivates such as catechol, 4-t-butyl-catechol, 3-methylcatechol, 3,4-dihydroxyphenylalanine, 3,4-dihydroxyphenylacetic acid, and 4-methoxyphenol are cited as capable of removing melanin from urine samples.
Certain metabolites of melanin serve as diagnostic markers or monitoring indices for melanoma. Jimbow et al. (Society for Investigative Dermatology, Inc. 100:No. 3, Supplement, March, 1993) have reported that the synthesis of pheomelanin is markedly increased in malignant melanoma and dysplastic nevi, that high levels of metabolites of pheomelanin and eumelanin can be detected in the blood and urine of patients with metastatic melanomas, that the release of melanin metabolites correlates with tumor thickness and tumor load including the extent of metastases, that synthesis of melanosomal proteins becomes aberrant in malignant melanoma, and that antigenic epitopes uniquely expressed in malignant melanoma can be identified.
Methylene blue, a phenothiazine derivative, is reported to have affinity for melanin and has been introduced to clinical trials for diagnosis and therapy.
U.S. Pat. No. 4,749,559 reportedly discloses a method of detecting melanin-containing matter by reacting melanin-containing matter with an enantiomer of 2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepin-7-ol (BZZ) and determining the binding of the enantiomer with melanin. The detecting requires that the enantiomer be labeled with a radioactive compound such as .sup.125 I. BZZ appears in the normal brain (caudate) at levels that may be toxic and appears significantly less tumor-specific than an ideal detecting agent.
U.S. Pat. No. 5,310,539 to Williams, incorporated herein by reference, reportedly provides melanin-based agents for image enhancement. Native or synthesized melanin is reported to act as a contrast agent by itself, however, incorporating a paramagnetic metal into the melanin polymer enhances significantly its ability to affect contrast in magnetic resonance imaging (MRI). Natural melanin is cited as possibly having some endogenous metals but such metals do not cause a relaxation effect similar to those provided by Williams. The high toxicity of gadolinium is cited as making it unlikely to be available for incorporation into a natural melanin produced in vivo. These melanin-based contrast agents, therefore, are not useful for detecting in vivo melanin, or melanin being assayed in an in vitro assay.
The challenge remains to develop low-cost, readily available, and highly sensitive, specific, laboratory tests to improve the diagnosis and monitoring of malignant melanotic melanoma. An ideal agent would also be capable of being stably labeled, and when used in vivo, would be nontoxic and quickly cleared from the system.
Texaphyrins are aromatic pentadentate macrocyclic "expanded porphyrins" useful as MRI contrast agents, as radiosensitizers, as chemosensitizers, and in photodynamic therapy. Texaphyrin is considered as being an aromatic benzannulene containing both 18p- and 22p-electron delocalization pathways. Texaphyrin molecules absorb strongly in the tissue-transparent 700-900 nm range, and they exhibit inherent selective uptake or biolocalization in certain tissues, particularly regions such as, for example, liver, atheroma or tumor tissue. Texaphyrins have exhibited significant tumor selectivity as detected by fluorescence and magnetic resonance imaging. Texaphyrins and water-soluble texaphyrins, method of preparation and various uses have been described in U.S. Pat. Nos. 4,935,498; 5,162,509; 5,252,720; 5,256,399; 5,272,142; 5,292,414; 5,369,101; 5,432,171; 5,439,570; 5,451,576; 5,457,183; 5,475,104 5,504,205; 5,525,325; 5,559,207; 5,565,552; 5,567,687; 5,569,759; 5,580,543; 5,583,220; 5,587,371; 5,587,463; 5,591,422; 5,594,136; 5,595,726; 5,599,923; 5,599,928; 5,601,802; 5,607,924; and 5,622,946; PCT publications WO 90/10633, 94/29316, 95/10307, 95/21845, 96/09315, 96/38461 and 96/40253; allowed U.S. patent applications Ser. Nos. 08/484,551, 08/591,318 and 08/624,311; and pending U.S. patent applications Ser. Nos. 08/458,347, 08/657,947, 08/700,277 and 08/763,451; each patent, publication, and application is incorporated herein by reference.
U.S. Food and Drug Administration phase I clinical trials of the texaphyrin photosensitizer, lutetium texaphyrin, for the photodynamic treatment of metastatic cancers involving the skin or subcutaneous tissue have been completed, and phase II testing is in progress. Gadolinium texaphyrin is currently in a multi-center phase Ib/II trial for the treatment of brain metastases.
The present invention overcomes cited problems with prior art methods of melanin and melanoma detection by providing texaphyrins or texaphyrin metal complexes as detection reagents for the presence of melanin and melanin metabolites that are predictive of the presence of melanotic melanoma.